Upon cell infection, Reoviridae capsids never completely disassemble, and viral transcripts are extruded from the icosahedral core particle. As a consequence the core must contain all the necessary enzymatic reactions for transcription and capping. The interactions between viral proteins and between these proteins and the viral genome in the core, is highly specific and the core may be considered to function as a precise molecular machine. Atomic structures for the cores of three members of this family (Bluetongue virus, Rice dwarf virus and mammalian orthoreovirus) are available, and are among the largest structures solved by X- ray diffraction. However, the core architecture of BTV (and RDV) is different with only reovirus having defined capping turrets. For BTV, capping protein is co-localised with the RNA-dependent-RNA-polymerase (RdRp) at the fivefold vertices of the core. Although the transcription complex within the core has been localised, it was not possible to resolve its structural details from the crystal structure. The objective of this application is to provide a complete structural and biochemical understanding of how the BTV transcription complex functions. Completion of this goal will pave the way for the rational design of inhibitors to block orbivirus replication and provide lead compounds for related viruses of pathogenic significance. Furthermore, the work will contribute to the growing areas of understanding the structural basis of transcription and for understanding how complex machines can be constructed from modular protein-based units. BTV is uniquely placed to deliver this information. It is the only non-turreted member of the Reoviridae for which an atomic core structure and complete in vitro activity of purified recombinant enzymatic proteins is available. The proposed research will build on these reagents and on our recent crystal structure of the BTV capping enzyme to understand the molecular basis of enzyme function and how the enzymatic proteins of the core act in concert to achieve transcription. In Specific Aim 1 we will undertake a detailed program of mutagenesis, biochemical and biophysical studies to understand the structure-function relationships of the four enzymatic activities of the capping enzyme. Specific Aim 2 will focus on the protein-protein interactions between the capping and RdRp proteins, and between these proteins and the major structural proteins of the core. Specific Aim 3 will focus on structure-function studies of the core associated viral helicase protein to understand if this protein is functional in transcription or packaging of the viral genome. Finally, in Specific Aim 4 recombinant proteins will be used to reconstitute viral transcription complexes in vitro to understand how these proteins interact with and affect the activity of each other. In all approaches we will use a combination of structural (NMR, cryo-EM, X-ray crystallography, dynamic and static light scattering) and biochemical (single molecule kinetic studies, mutagenesis, Raman and IR spectroscopy, HPLC, enzyme assays to follow kinetics, surface plasmon resonance) approaches. PUBLIC HEALTH RELAVANCE: The project aims to provide a complete understanding of how a complex virus functions as a nanomachine. Completion of this goal will pave the way for the rational design of inhibitors to block virus (in particular, an animal virus) replication and provide lead compounds for related viruses of pathogenic significance to humans and animals.